Antiviral compounds

ABSTRACT

This invention relates to purine compounds of formula (I):  
                 
 
     R 1  is NH 2  or OH; R 2  is H or NH 2 ; R 3  is H or alkyl; each of m and n, independently, is 1, 2, 3, or 4; X is O, S, or NH; and Y is H, halogen, OR a , P(O)(OR a ) 2 , or P(O)(OR a )(OR b ), in which R a  is H, alkyl, aryl, heteroaryl, cyclyl, heterocyclyl, and R b  is  
                 
 
     wherein A is adenine, guanine, cytosine, uracil, or thymine; R c  is H or OH; R d  is H or alkyl; R e  is H, alkyl, or 5-ethylidene-(3,4-dialkoxyl)-furan-2-one; provided that if R 1  is NH 2 , R 2  is H; and if R 1  is OH, R 2  is NH 2 .

BACKGROUND

[0001] The DNA genome in the particle of a DNA virus can bedouble-stranded, single-stranded, or partially double-stranded DNA.Papovaviridae (e.g., papillomaviruses), herpesviridae (e.g., herpessimplex viruses), and adenoviridae (e.g., adenoviruses) containdouble-stranded DNA genomes. Some viruses from parvoviridae (e.g.,parvoviruses) contain single-stranded DNA as their genomes. Some virusesfrom hepadnaviridae (e.g., hepatitis B virus) contain partiallydouble-stranded DNA as their genomes, and replicate their genomesthrough RNA intermediates. In contrast, retroviruses are a family of RNAviruses that replicate through a DNA intermediate. Examples ofretroviruses include Moloney murine sarcoma viruses, human T-celllymphotrophic viruses, human immunodeficiency viruses, and human foamyviruses.

[0002] Viruses described above cause a variety of diseases, such as theflu, common cold, herpes, measles, small pox, and encephalitis.Vaccination only offers protection for uninfected individuals for a fewviral diseases. Thus, there is a need for identifying therapeutic agentsuseful for preventing or treating viral infection.

SUMMARY

[0003] The present invention is based, in part, on the discovery ofnucleotide analogs that possess anti-viral activity.

[0004] In one aspect, this invention relates purine compounds of formula(I):

[0005] R₁ is NH₂ or OH; R₂ is H or NH₂; R₃ is H or alkyl; each of m andn, independently, is 1, 2, 3, or 4; X is O, S, or NH; and Y is H,halogen, OR^(a), P(O)(OR^(a))₂, or P(O)(OR^(a))(OR^(b)), in which R^(a)is H, alkyl, aryl, heteroaryl, cyclyl, heterocyclyl, and R^(b) is

[0006] (referred to as “sugar-A(R^(c))-OP(O)(OR^(d))(OR^(e))”hereinafter), wherein A is adenine, guanine, cytosine, uracil, orthymine; R^(c) is H or OH; R^(d) is H or alkyl; R^(e) is H, alkyl, or5-ethylidene-(3,4-dialkoxyl)-furan-2-one; provided that if R₁ is NH₂, R₂is H; and if R₁ is OH, R₂ is NH₂. Note that the left atom shown in anyof the substituted groups set forth above is closest to the purine ring.

[0007] Referring to formula (I), a subset of the purine compounds arethose in which each of R₁ is NH₂ and R₂ is H. In these compounds, R₃ canbe H; X can be O; and each of m and n, independently, is 1 or 2. In someembodiments, m is 1, X is O, n is 2, and Y is OR^(a), in which R^(a) canbe H. In other embodiments, m is 2, X is O, n is 1, and Y isP(O)(OR^(a))₂, in which R^(a) can be H. In still other embodiments, m is2, X is O, n is 1, and Y is P(O)(OR^(a))(OR^(b)), in which R^(a) can beH, and R^(b) can be sugar-A(R^(c))-OP(O)(OR^(d))(OR^(e)).

[0008] Another subset of the purine compounds of formula (I) are thosein which R₁ is OH and R₂ is NH₂. In these compounds, R₃ can be H; X canbe O; and each of m and n, independently, is 1 or 2. In someembodiments, m is 1, X is O, n is 2, and Y is OR^(a), in which R^(a) canbe H. In other embodiments, m is 2, X is O, n is 1, and Y isP(O)(OR^(a))₂, in which R^(a) can be H. In still other embodiments, m is2, X is O, n is 1, and Y is P(O)(OR^(a))(OR^(b)), in which R^(a) can beH, and R^(b) can be sugar-A(R^(c))-OP(O)(OR^(d))(OR^(e)).

[0009] In another aspect, this invention encompasses purine compounds offormula (II):

[0010] R₁ is NH₂ or OH; R₂ is H or NH₂; R₃ is H or alkyl; each of m andn, independently, is 1, 2, 3, or 4; X is O, S, or NH; and Y isP(O)(OR^(a))(OR^(b)), in which R^(a) is H, alkyl, aryl, heteroaryl,cyclyl, heterocyclyl; and R^(b) is5-ethylidene-(3,4-dialkoxy)-furan-2-one orsugar-A(R^(c))-OP(O)(OR^(d))(OR^(e)); wherein A is adenine, guanine,cytosine, uracil, or thymine; R^(c) is H or OH; R^(d) is H or alkyl;R^(e) is H, alkyl, or 5-ethylidene-(3,4-dialkoxyl)-faran-2-one; providedthat if R₁ is NH₂, R₂ is H; and if R₁ is OH, R₂ is NH₂.

[0011] Referring to formula (II), a subset of the purine compounds arethose in which each of R₁ is NH₂ and R₂ is H. In these compounds, R₃ canbe H; X can be O; and each of m and n, independently, is 1 or 2. In someembodiments, m is 2, X is O, n is 1, and Y is P(O)(OR^(a))(OR^(b)), inwhich R^(a) can be H, and R^(b) can be5-ethylidene-(3,4-dialkoxy)-furan-2-one.

[0012] Another subset of the purine compounds of formula (II) are thosein which R₁ is OH and R₂ is NH₂. In these compounds, R₃ can be H; X canbe O; and each of m and n, independently, is 1 or 2. In someembodiments, m is 2, X is O, n is 1, and Y is P(O)(OR^(a))(OR^(b)), inwhich R^(a) can be H, and R^(b) can be5-ethylidene-(3,4-dialkoxy)-furan-2-one.

[0013] Also within the scope of this invention is a method of preparingcertain purine compounds of formula (I). The method includes reacting acompound of formula (III):

[0014] with an alkyl-X-(CH₂) halide to obtain a compound of formula(IV):

[0015] In formulae (III) and (IV), R₁ is NH₂ or OH; R₂ is H or NH₂; R₃is H or alkyl; and X is O, S, or NH; provided that if R₁ is NH₂, R₂ isH; and if R₁ is OH, R₂ is NH₂.

[0016] Unless specifically pointed out, alkyl, aryl, heteroaryl, cyclyl,heterocyclyl, adenine, guanine, cytosine, uracil, and thymine mentionedabove include both substituted and unsubstituted moieties. The term“substituted” refers to one or more substituents (which may be the sameor different), each replacing a hydrogen atom. Examples of substituentsinclude, but are not limited to, halogen, cyano, nitro, hydroxyl, amino,mercapto, C₁˜C₆ alkyl, C₁˜C₆ alkenyl, C₁˜C₆ alkynyl, aryl, heteroaryl,C₄˜C₈ cyclyl, C₄˜C₈ heterocyclyl, alkyloxy, aryloxy, alksulfanyl,arylsulfanyl, alkylamino, arylamino, dialkylamino, diarylamino,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, alkylcarboxyl,arylcarboxyl, heteroarylcarboxyl, alkyloxycarbonyl, aryloxycarbonyl,heteroaryloxycarbonyl, alkylcarbamido, arylcarbamido, heterocarbamido,alkylcarbamyl, arylcarbamyl, heterocarbamyl, wherein each of alkyl(including alk), alkenyl, aryl, heteroaryl, cyclyl, and heterocyclyl isoptionally substituted with halogen, cyano, nitro, hydroxyl, amino,mercapto, C₁˜C₆ alkyl, aryl, heteroaryl, alkyloxy, aryloxy,alkylcarbonyl, arylcarbonyl, alkylcarboxyl, arylcarboxyl,alkyloxycarbonyl, or aryloxycarbonyl.

[0017] The term “alkyl” refers to both linear and branched alkyl. Theterm “cyclyl” refers to a hydrocarbon ring containing 4 to 8 carbons.The term “heterocyclyl” refers to a ring containing 4 to 8 ring membersthat have at least one heteroatom (e.g., S, N, or O) as part of thering. The term “aryl” refers to a hydrocarbon ring system having atleast one aromatic ring. Examples of aryl moieties include, but are notlimited to, phenyl, naphthyl, and pyrenyl. The term “heteroaryl” refersto a hydrocarbon ring system having at least one aromatic ring whichcontains at least one heteroatom such as O, N, or S. Examples ofheteroaryl moieties include, but are not limited to, furyl, pyrrolyl,thienyl, oxazolyl, imidazolyl, thiazolyl, pyridinyl, pyrimidinyl,quinazolinyl, and indolyl.

[0018] All of the purine compounds described above include the compoundsthemselves, as well as their salts. The salts, for example, can beformed between a positively charged substituent (e.g., amino) on acompound and an anion. Suitable anions include, but are not limited to,chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate,methanesulfonate, trifluoroacetate, and acetate. Likewise, a negativelycharged substituent (e.g., carboxylate) on a compound can form a saltwith a cation. Suitable cations include, but are not limited to, sodiumion, potassium ion, magnesium ion, calcium ion, and an ammonium cationsuch as teteramethylammonium ion.

[0019] In addition, some of the just-described purine compounds have oneor more double bonds, or one or more asymmetric centers. Such compoundscan occur as racemates, racemic mixtures, single enantiomers, individualdiastereomers, diastereomeric mixtures, and cis- or trans- or E- or Z-double bond isomeric forms.

[0020] Exemplary purine compounds of formula (I) and (II) include:

[0021] This invention also features a method of treating infection byvirus. The method includes administering to a subject in need thereof aneffective amount of a purine compound of formula (I) or formula (II) asdescribed above. Examples of the viruses include DNA viruses such asherpesviridae (e.g., herpes simplex viruses) and retroviruses such asMoloney murine sarcoma viruses and human immunodeficiency viruses (e.g.,human immunodeficiency viruses-1 or -2).

[0022] As used herein, the term “treating infection” refers to use ofone or more purine compounds described above for preventing or treatinginfection by virus, or other disease states secondary to viralinfection, e.g., cervical cancer induced by Papilovirus.

[0023] Also within the scope of this invention are a compositioncontaining one or more of the aforementioned purine compounds for use intreating viral infection, and the use of such a composition for themanufacture of a medicament for infection treatment.

[0024] Other features or advantages of the present invention will beapparent from the following detailed description of several embodiments,and also from the appending claims.

DETAILED DESCRIPTION

[0025] This invention relates to the purine compounds of formula (I) andformula (II) as described in the Summary section.

[0026] As shown in Scheme la below, the purine compounds of formula (I)can be prepared by a novel procedure: alkylation of N⁹-tritylated purinecompounds (III), followed by concomitant self-detritylation to yield thedesired N⁷-alkylated purine nucleosides (IV). In this Scheme, R₁, R₂,R₃, and X are defined in the Summary section.

[0027] Compound (III) can be obtained by silylation of a purine (e.g.,guanine) with hexamethyldisilazane in the presence of a catalytic amountof (NH₄)₂SO₄ at an elevated temperature, followed by condensation of theresultant silylated purine with trityl chloride.

[0028] Other than the just-described procedure, the purine compounds offormula (I) and formula (II) can be prepared by methods well known inthe art. Scheme 1b shown below adepicts synthesis of the compounds offormula (I). In this scheme, R₁, R₂, R₃, m, n, X, and Y are defined inthe Summary section.

[0029] More specifically, a purine compound is alkylated with3-bromopropionitrile in the presence of NaH to give N⁹-cyanoethyl purineintermediate. Reaction of this intermediate with a methyliodo-ester andlithium 2,2,6,6-tetramethylpiperidine affords a mixture of N⁷-alkylatedand N⁹-alkylated ester-containing products. Reduction of the ester inN⁹-alkylated product gives another intermediate, which is converted to adesired purine compound of formula (I) by an alkylation reaction or byreacting with a phosphonate in the presence of tert-butoxide. Similarly,reduction of the ester in N⁷-alkylated product gives a compound, whichreacts with a phosphonate to produce an N⁷-substitutedphosphonate-purine.

[0030] Reaction of the N⁷-substituted phosphonate-purine with a halidein the presence of NaHCO₃ affords a desired compound of formula (II).

[0031] Scheme 1c below depicts synthesis of the compounds of formula(II). In this scheme, R₁, R₂, R₃, R^(a), R^(b), m, n, X, and Y aredefined in the Summary section.

[0032] The chemicals used in the above-described synthetic routes mayinclude, for example, solvents, reagents, catalysts, protecting groupand deprotecting group reagents.

[0033] The methods described above may also additionally include steps,either before or after the steps described specifically herein, to addor remove suitable protecting groups in order to ultimately allowsynthesis of the purine compound. In addition, various synthetic stepsmay be performed in an alternate sequence or order to give the desiredcompounds. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizingapplicable purine compounds are known in the art and include, forexample, those described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2^(nd) Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995) and subsequent editions thereof.

[0034] A purine compound thus synthesized can be further purified by amethod such as column chromatography, high pressure liquidchromatography, or recrystallization.

[0035] Also within the scope of this invention is a pharmaceuticalcomposition that contains an effective amount of at least one purinecompound of the present invention and a pharmaceutically acceptablecarrier. Further, this invention covers a method of administering to asubject in need of treating viral infection an effective amount of oneor more of the purine compounds. The term “treating” is defined as theapplication or administration of a composition including the purinecompound to a subject, who has a viral infection, a symptom of theinfection, a disease or disorder secondary to the infection, or apredisposition toward the infection, with the purpose to cure,alleviate, relieve, remedy, or ameliorate the infection, the symptom ofthe infection, the disease or disorder secondary to the infection, orthe predisposition toward the infection. “An effective amount” isdefined as the amount of the purine compound which, upon administrationto a subject in need thereof, is required to confer therapeutic effecton the subject. An effective amount of the purine compound may rangefrom 5 mg/Kg to 20 mg/Kg. Effective doses also vary, as recognized bythose skilled in the art, depending on route of administration,excipient usage, and the possibility of co-usage with any othertherapeutic agent, such as an antiviral agent.

[0036] To practice the method of the present invention, theabove-described pharmaceutical composition can be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrastemal, intrathecal, intralesional and intracranial injection orinfusion techniques.

[0037] A sterile injectable composition, e.g., a sterile injectableaqueous or oleaginous suspension, can be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as Tween 80) and suspending agents. The sterile injectablepreparation can also be a sterile injectable solution or suspension in anon-toxic parenterally acceptable diluent or solvent, for example, as asolution in 1,3-butanediol. Among the acceptable vehicles and solventsthat can be employed are mannitol, water, Ringer's solution and isotonicsodium chloride solution. In addition, sterile, fixed oils areconventionally employed as a solvent or suspending medium (e.g.,synthetic mono- or di-glycerides). Fatty acids, such as oleic acid andits glyceride derivatives are useful in the preparation of injectables,as are natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions can also contain a long-chain alcohol diluentor dispersant, or carboxymethyl cellulose or similar dispersing agents.Other commonly used surfactants such as Tweens or Spans or other similaremulsifying agents or bioavailability enhancers which are commonly usedin the manufacture of pharmaceutically acceptable solid, liquid, orother dosage forms can also be used for the purposes of formulation.

[0038] A composition for oral administration can be any orallyacceptable dosage form including, but not limited to, capsules, tablets,emulsions and aqueous suspensions, dispersions and solutions. In thecase of tablets for oral use, carriers which are commonly used includelactose and corn starch. Lubricating agents, such as magnesium stearate,are also typically added. For oral administration in a capsule form,useful diluents include lactose and dried corn starch. When aqueoussuspensions or emulsions are administered orally, the active ingredientcan be suspended or dissolved in an oily phase combined with emulsifyingor suspending agents. If desired, certain sweetening, flavoring, orcoloring agents can be added. A nasal aerosol or inhalation compositioncan be prepared according to techniques well-known in the art ofpharmaceutical formulation and can be prepared as solutions in saline,employing benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons, and/or othersolubilizing or dispersing agents known in the art. A purinecompound-containing composition can also be administered in the form ofsuppositories for rectal administration.

[0039] The carrier in the pharmaceutical composition must be“acceptable” in the sense of being compatible with the active ingredientof the formulation (and preferably, capable of stabilizing it) and notdeleterious to the subject to be treated. For example, solubilizingagents such as cyclodextrins, which form specific, more solublecomplexes with the indole compounds, or one or more solubilizing agents,can be utilized as pharmaceutical excipients for delivery of the purinecompounds. Examples of other carriers include colloidal silicon dioxide,magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow#10.

[0040] A purine compound of this invention can be preliminarily screenedfor its efficacy in treating viral infection by one or more of thefollowing in vitro assays.

[0041] In one assay, a purine compound is tested for its inhibition ofcytopathogenicity against the herpes simplex type 1 virus, herpessimplex simplex type 2 virus, thymidine kinase-positive and thymidinekinase-deficient strains of varicell-zoster virus, or humancytomegalovirus in Vero cells. The method for measuring viruses-inducedcytophogenicity in Vero cell cultures, as well as the toxicity of thetest compound toward HeLa and Vero cells, has been described in e.g., DeClercq et al. (1980) J. Infect. Dis. 141: 563-574.

[0042] In another assay, a purine compound is tested for its inhibitionof cytopathogenicity against the human immunodeficiency viruses HIV-1(IIIB) and HIV-2 (LAV-2) in MT4 cells. The method for measuringviruses-induced cytophogenicity in MT4 cells or CEM cells, as well asthe toxicity toward MT4 and CEM cells, has been described in e.g.,Averett, (1989) J. Virol. Methods 23: 263-276.

[0043] The antiviral activity of a purine compound can be furtherassessed using an in vivo animal model. See the specific examples below.

[0044] Without further elaboration, it is believed that the abovedescription has adequately enabled the present invention. The followingspecific embodiments are, therefore, to be construed as merelyillustrative, and not limitative of the remainder of the disclosure inany way whatsoever. All of the publications cited herein are herebyincorporated by reference in their entirety.

[0045] General. For anhydrous reactions, glassware was dried overnightin an oven at 120° C. and cooled in a desiccator over anhydrous CaSO₄ orsilica gel. Reagents were purchased from Fluka and enzymes from SigmaChemical Co. Solvents, including dry ether and tetrahydrofuran (THF),were obtained by distillation from the sodium ketyl of benzophenoneunder nitrogen. Other solvents, including chloroform, dichloromethane,ethyl acetate, and hexanes were distilled over CaH₂ under nitrogen.Absolute methanol and ethanol were purchased from Merck and used asreceived.

[0046] Melting points were obtained with a Büchi 510 melting pointapparatus. Infrared (IR) spectra were recorded on a Beckman IR-8spectrophotometer. The wavenumbers reported are referenced to the 1601cm⁻¹ absorption of polystyrene. Proton NMR spectra were obtained on aVarian XL-300 (300 MHz) Spectrometer. Chloroform-d anddimethylsulfoxide-d₆ were used as solvent; Me₄Si (δ 0.00 ppm) was usedas an internal standard. All NMR chemical shifts are reported as 6values in parts per million (ppm) and coupling constants (J) are givenin hertz (Hz). The splitting pattern abbreviations are as follows: s,singlet; d, doublet; t, triplet; q, quartet; br, broad; m, unresolvedmultiplet due to the field strength of the instrument; and dd, doubletof doublets. UV spectroscopy was carried out using an HP8452A diodearray spectrophotometer. Mass spectra were carried out on a VG 70-250 Smass spectrometer. Microanalyses were performed on a Perkin-Elmer 240-Bmicroanalyzer.

[0047] Purification on silica gel refers to gravity columnchromatography on Merck Silica Gel 60 (particle size 230-400 mesh).Analytical TLC was performed on precoated plates purchased from Merck(Silica Gel 60 F₂₅₄). Compounds were visualized by use of UV light, I₂vapor, or 2.5% phosphomolybdic acid in ethanol with heating.

[0048] 9-[(2-Hydroxyethoxy)methyl]adenine 1,9-[(2-hydroxyethoxy)methyl]guanine (acyclovir 2),9-(β-D-arabinofaranosyl)adenine (ara-A 3),9-[2-(phosphonomethoxy)ethyl]adenine (PMEA 4) were used for comparisonin Examples 8-10. Compounds 9, 14, 20, 24, 25, 28, and 29 were preparedby the methods described in Examples 1-7.

EXAMPLE 1 Synthesis of Compound 9

[0049]

[0050] As shown in Scheme 2, N⁹-tritylated guanine 6 was synthesized intwo steps. Silylation of guanine 5 with hexamethyldisilazane (HMDS) inthe presence of a catalytic amount of (NH₄)₂SO₄ at refluxing temperaturefollowed by condensation of the resultant silylated guanine with tritylchloride in MeCN at 25° C. afforded the desired N⁹-tritylated guanine 6in 90% yield. Treatment of 6 with (chloroethoxy)methyl chloride(Hakimelahi & Khalafi-Nezhad (1989) Helv. Chim. Acta 72: 1495-1550) inDMF at room temperature gave the corresponding N⁷-alkylated guanine 7 in88% yield. Likewise, treatment of 6 with[2-(p-methoxyphenyloxy)ethoxy]methyl chloride (Khorshidi (1986) DoctoralThesis in Pharmacy, Faculty of Medicine, Isfahan University, Isfahan,Iran) led to the N⁷-isomer 8 in 82% yield. Removal of thep-methoxyphenyl moiety was then achieved by treatment with cericammonium nitrate (CAN, Fukuyama et al. (1985) Tetrahedron Lett. 26:6291-6292) in a mixture of MeCN and H₂O (3:1) at 0-25° C. to affordcompound 9 in 70% yield. For compound 9 (i.e., a N⁷-isomer) and itscorresponding N⁹-isomer, their ¹H and ¹³C NMR spectra are different.See, e.g., Kjellberg & Johansson (1986) Tetrahedron 42: 6541-6544;Shiragami et al. (1995) Nucleosides & Nucleotides 14: 337-340; andBailey & Hamden (1987) Nucleosides & Nucleotides 6: 555-574. The ¹Hsignals of H₂C(1′) (5.81 ppm) and HC(8) (8.67 ppm) for the N⁷-isomerwere found to be shifted downfield relative to the corresponding signalsof the N⁹-isomer, in which the H₂C(1′) and HC(8) resonated at 5.35 and7.81 ppm, respectively. On the other hand, the ¹H signals for NH₂ wasobserved to be shifted upfield for the N⁷-isomer, 5.96 ppm for theN⁷-isomer, relative to the corresponding signal for N⁹-isomer, which wasobserved at 6.52 ppm. The ¹³C NMR signals for C(1′) (75.25 ppm) and C(8)(143.92 ppm) of the N⁷-isomer were found to be shifted downfieldrelative to the corresponding signals of the N⁹-isomer, which wereobserved respectively at 71.64 and 137.89 ppm. In contrast, the signalof C(5) of the N⁷-isomer resonated at 107.16 ppm, which was upper fieldto that of the N⁹-isomer at 116.52 ppm. The UV λ_(max) of the N⁷-isomerappeared at 289 nm, whereas the corresponding λ_(max) of the N⁹-isomerappeared at 253 and 273 (sh) nm.

[0051] 9-(Triphenylmethyl)guanine 6. Guanine 5 (1.51 g, 9.99 mmol) and(NH₄)₂SO₄ (100 mg) were suspended in HMDS (150 mL) and refluxed for 24h. The solvent was evaporated under reduced pressure and the residue wasdissolved in CH₃CN (150 mL). Triphenylmethyl chloride (2.79 g, 10.0mmol) was added and the reaction mixture was stirred at 25° C. for 7.0h. The solution was concentrated under reduced pressure and the residuewas purified by use of column chromatography (hexanes/EtOAc=1.5:8.5) toafford 6 (3.54 g, 8.99 mmol) in 90% yield: mp 268-270° C.;R_(f)(hexanes/EtOAc=1:2) 0.34; UV (EtOH) λ_(max) 254 (ε 13,870), 278(sh); ¹H NMR (DMSO-d₆) δ 5.97 (s, 2 H, NH₂), 7.09-7.39 (m, 16H,HC₈+C(C₆H₅)₃), 10.45 (br s, 1H, NH); MS m/z 393 (M⁺). Anal. (C₂₄H₁₉N₅O)C, H, N; calcd (%): 73.26, 4.87, 17.80; found (%): 73.20, 4.81, 17.78.

[0052] 7-[(2-Chloroethoxy)methy]guanine 7. To a solution of 6 (1.77 g,4.49 mmol) in DMF (30 mL) was added (2-chloroethoxy)methyl chloride(0.65 g, 5.0 mmol). The reaction mixture was stirred at 25° C. for 8.0h. The solution was then partitioned between EtOAc (100 mL) and water(100 mL). The EtOAc solution was washed with water (4×100 mL); then itwas dried over MgSO₄ (s) and filtered. Evaporation under reducedpressure and purification of the residue by use of column chromatography(EtOAc) afforded 7 (1.07 g, 4.39 mmol) in 88%: mp>280° C. (dec.);R_(f)(hexanes/EtOAc=1:2) 0.20; UV (EtOH) λ_(max) 288 (ε 15,100); ¹H NMR(DMSO-d₆) δ 3.67 (t, J=6.10 Hz, 2H, CH₂Cl), 3.75 (t, J=6.10 Hz, 2H,OCH₂), 5.62 (s, 2H, H₂C_(1′)), 6.15 (s, 2H, NH₂), 7.58 (s, 1H, NH), 8.16(s, 1H, HC₈); ¹³C NMR (DMSO-d₆) δ 43.39 (CH₂Cl), 68.50 (OCH₂) 74.71(C_(1′)), 107.71 (C₅), 143.87 (C₈), 153.15 (C₄), 154.37 (C₂), 159.12(C₆); MS m/z 243 (M⁺, Cl-cluster). Anal (C₈H₁₀ClN₅O₂) C, H, N, Cl; calcd(%): 39.43, 4.14, 28.74, 14.56; found (%): 39.38, 4.13, 28.70, 14.45.

[0053] 7-[(2-(p-Methoxyphenyloxy)ethoxy)methyl]guanine 8. Compound 8(2.72 g, 8.20 mnmol) was prepared in 82% yield from 6 (3.64 g, 9.25mmol) and (2-(p-methoxyphenyloxy)ethoxy)methyl chloride (2.18 g, 10.1mmol) in DMF (100 mL) by the method used for the synthesis of 7: mp>250°C. (dec.); R_(f)(hexanes/EtOAc=1:2) 0.18; UV (EtOH) λ_(max) 290 (ε16,500); ¹H NMR (DMSO-d₆) δ 3.70 (s, 3H, OCH₃), 4.02-4.10 (m, 4H,O(CH₂)₂O), 5.74 (s, 2H, H₂C_(1′)), 5.88 (s, 2H, NH₂), 6.67, 6.70(AA′BB′, J=9.30 Hz, 4H, C₆H₄), 7.50 (s, 1H, NH), 8.85 (s, 1H, HC₈); ¹³CNMR (DMSO-d₆) δ 56.12 (CH₃), 68.74 (OCH₂), 70.20 (CH₂Oph),75.31(C_(1′)), 107.25 (C₅), 144.02 (C₈), 153.25 (C₄ 0, 154.48 (C₂),159.26 (C₆), 115.55, 116.38, 153.51, 155.56 (C₆H₄); MS m/z331 (M⁺). Anal(C₁₅H₁₇N₅O₄) C, H, N; calcd (%): 54.37, 5.17, 21.13; found (%): 54.26,5.20, 21.19.

[0054] 7-[(2-Hydroxyethoxy)methyl]guanine 9. To a solution of 8 (1.36 g,4.10 mmol) in a mixture of CH₃CN (30 mL) and water (10 mL) was added CAN(2.25 g, 4.10 mmol) at 0° C. The stirred reaction mixture was allowed towarm-up to 25° C. within 1.0 h. Water (30 mL) was added to afford asolid. Filtration and crystallization of the solid from EtOH/water (4:1)gave 9 (0.65 g, 2.88 mmol) in 70% yield: mp>280° C. (dec.);R_(f)(hexanes/EtOAc=1:2) 0.08; UV (EtOH) λ_(max) 289 (ε 16,000); ¹H NMR(DMSO-d₆) δ 3.61-3.82 (m, 4H, O(CH₂)₂O), 4.85 (br s, 1H, OH), 5.81 (s,2H, H₂C_(1′)), 5.96 (br s, 2H, NH₂), 6.61 (s, 1H, NH), 8.67 (s, 1H,HC₈); ¹³C NMR (DMSO-d₆) δ 60.91 (CH₂OH), 70.01 (OCH₂) 75.25 (C_(1′)),107.16 (C₅), 143.92 (C₈), 153.20 (C₄), 154.40 (C₂), 159.30 (C₆); MS m/z225 (M⁺). Anal (C₈H₁₁N₅O₃) C, H, N; calcd (%): 42.66, 4.92, 31.10; found(%): 42.75, 4.84, 31.21.

EXAMPLE 2 Synthesis of Compound 14

[0055]

[0056] As shown in Scheme 3, by the same synthetic strategy shown inScheme 2, novel N⁷-alkylated adenines 12 and 14 were obtained fromadenine 10 via the N⁹-tritylated adenine 11. Reaction of 11 with(2-chloroethoxy)methyl chloride in CH₂Cl₂ at 25° C. gave N⁷-adeninederivative 12 in 90% yield. Likewise, reaction of 11 with[2-(p-methoxyphenyloxy)ethoxy]methyl chloride in CH₂Cl₂ at 25° C. gaveN⁷-isomer 13 in 86% yield. Treatment of 13 with CAN then produced thedeprotected compound 14 in 80% yield.

[0057] For compound 14 (i.e., a N⁷-isomer) and its correspondingN⁹-isomer, their ¹H and ¹³C NMR spectra are different. In methanol-d₄,pronounced downfield shifts were observed for the signals resulting fromthe H₂C(1′) (5.79 ppm) and HC(8) (8.60 ppm) of the N⁷-isomer whencompared with those of the N⁹-isomer, respectively observed at 5.67 and8.27 ppm. In DMSO-d₆, the NH₂ signals of the 187-isomer was shifteddownfield to ˜9.2 and 110.0 ppm, whereas the corresponding signals ofthe N⁹-isomer appeared as a broad peak at ˜7.3 ppm. The ¹³C NMR signalsfor C(1′) (82.01 ppm) and C(8) (147.68 ppm) for the N⁷-isomer were foundto be shifted downfield relative to those of the N⁹-isomer which wereobserved respectively at 74.31 and 143.14 ppm. The signals of the C(2)(144.29 ppm) and C(6) (152.12 ppm) for the N⁷-isomer, however, werefound to be shifted upfield relative to those of the N⁹-isomer, observedrespectively at 153.74 and 157.19 ppm. Furthermore, the H—C(2) couplingconstants for the N⁷- and N⁹-isomers were respectively 220 and 204 Hz,whereas the corresponding H—C(8) coupling constants were respectively224 and 217 Hz. The attachment of the side chain at the N-7 position ofadenine was confirmed by HMQC spectroscopy, in which the H₂C(1′) andC(6) exhibited a strong interaction in the N⁷-isomer whereas thecorresponding N⁹-isomer showed long-range coupling between H₂C(1′) andC(4).

[0058] 9-(Triphenylmethyl)adenine 11. To a solution of 10 (1.35 g, 9.99mmol) and DMF (10 mL) in pyridine (30 mL) was added Ph₃Cl (2.79 g, 10.0mmol). The reaction mixture was stirred at 25° C. for 7.0 h. It was thendiluted with EtOAc (150 mL) and water (100 mL). The organic layer wasseparated and then washed with water (4×100 mL). It was dried over MgSO₄(s) and concentrated under reduced pressure to yield a foam.Purification was carried out by use of column chromatography(EtOAc/hexanes=8:2) to afford compound 11 (3.58 g, 9.48 mmol) in 95%yield: mp 260-262° C.; R_(f)(hexanes/EtOAc=1:2) 0.38; UV (EtOH) λ_(max)260 (ε 14,600); ¹H NMR (CDCl₃) δ 5.60 (s, 2H, NH₂), 7.15-7.31 (m, 15H,C(C₆H₅)₃), 7.74 (s, 1H, HC₂), 8.05 (s, 1H, HC₈); MS m/z 377 (M⁺ 0; Anal.(C₂₄H₁₉N₅) C, H, N; calcd. (%): 76.37, 5.07, 18.55; found (%): 76.39,5.16, 18.60.

[0059] 7-[(2-Chloroethoxy)methy]adenine 12. To a solution of 11 (1.79 g,4.74 mmol) in CH₂Cl₂ (70 mL) was added (2-chloroethoxy)methyl chloride(0.65 g, 5.0 mmol). The reaction mixture was stirred at 25° C. for 8.0 hto afford a solid. Filteration and crystallization from MeOH gave 12(1.66 g, 4.27 mmol) in 90% yield: mp 184-186° C.; R_(f)(hexanes/EtOAc=1:2) 0.28; UV (EtOH) λ_(max) 267 (ε 14,600); ¹H NMR(DMSO-d₆) δ 3.73 (t, J=4.5 Hz, 2H, CH₂Cl), 3.86 (t, J=4.5 Hz, 2H, OCH₂),5.76 (s, 2H, H₂C_(1′)), 8.49 (s, 1H, HC₂), 8.74 (s, 1H, HC₈), 9.32,10.05 (2 br, 2H, NH₂); ¹³C NMR (DMSO-d₆) δ 43.13 (CH₂Cl), 68.82 OCH₂78.10 (C_(1′)), 118.20 (C₅), 143.53 (C₂), 146.50 (C₈), 149.39 (C₄),151.60 (C₆); MS m/z 227 (M⁺, Cl-cluster). Anal. (C₈H₁₀ClN₅O) C, H, N,Cl; calcd (%): 42.20, 4.43, 30.76, 15.59; found (%): 42.32, 4.50, 30.69,15.62.

[0060] 7-[(2-(p-Methoxyphenyloxy)ethoxy)methyl]adenine 13. After 10 h,compound 13 (2.79 g, 8.86 mmol) was synthesized in 86% yield from 11(3.90 g, 10.3 mmol) and (2-(p-methoxyphenyloxy)ethoxy)methyl chloride(2.25 g, 10.4 mmol) in CH₂Cl₂ (100 mL) by the method used for thesynthesis of 12: mp 129-130° C.; R_(f)(hexanes/EtOAc=1:2) 0.21; UV(EtOH) λ_(max) 270 (ε 15,100); ¹H NMR (CD₃OD) δ 3.68 (s, 3H, OCH₃), 4.04(br s, 4H, O(CH₂)₂O), 5.83 (s, 2H, H₂C_(1′)), 6.58, 6.68 (AA′BB′, J=9.0Hz, 4H, C₆H₄), 8.29 (s, 1H, HC₂), 8.61 (s, 1H, HC₈); ¹³C NMR (CD₃OD) δ56.03 (CH₃), 68.70 (OCH₂), 70.18 CH₂OPh 82.27 (C_(1′)), 119.59 (C₅),144.17 (C₂), 147.57 (C₈), 150.25 (C₄), 151.97 (C₆), 115.50, 116.35,153.44, 155.53 (C₆H₄); MS m/z 315 (M⁺). Anal. (C₁₅H₁₇N₅O₃) C, H, N;calcd (%): 57.13, 5.43, 22.21; found (%): 57.24, 5.49, 22.34.

[0061] 7-[(2-Hydroxyethoxy)methyl]adenine 14. Compound 14 (0.74 g, 3.54mmol) was prepared in 80% yield from 13 (1.39 g, 4.43 mmol) and CAN(2.43 g, 4.43 mmol) in CH₃CN (30 mL) and water (10 mL) by the methodused for the synthesis of 9: mp 122-123° C.; R_(f)(hexanes/EtOAc=1:2)0.12; UV (EtOH) λ_(max) 265 (ε 14,800); ¹H NMR (CD₃OD) δ 3.69-3.79 (m,4H, O(CH₂)₂O), 5.79 (s, 2H, H₂C_(1′)), 8.32 (s, 1H, HC₂), 8.60 (s, 1H,HC₈); ¹³C NMR (CD₃OD) δ 61.80 (CH₂OH), 72.27 (OCH₂) 82.01 (C_(1′)),119.62 (C₅), 144.29 (C₂), 147.68 (C₈), 150.32 (C₄), 152.12 (C₆); MS m/z209 (M⁺). Anal (C₈H₁₁N₅O₂) C, H, N; calcd (%): 45.93, 5.30, 33.48; found(%): 45.80, 5.45, 33.51.

EXAMPLE 3 Synthesis of Compound 20.

[0062]

[0063] As shown in Scheme 4, alkylation of adenine 10 with3-bromopropionitrile in the presence of NaH in DMF gaveN⁹-(cyanoethyl)adenine 15 in 75% yield. Reaction of 15 with methyliodoacetate and lithium 2,2,6,6-tetramethylpiperidine (Li TMP) in THFafforded a mixture of N⁷-alkylated product 16 (60% yield) and N⁹-isomer17 (20% yield). Reduction of the ester group in 16 with NaBH₄ in wetTHF¹³ gave N⁷-(hydroxyethyl)adenine 18 in 55% yield. Conversion of 18 tophosphonate 19 (60% yield) was accomplished by use of diethyl(p-toluenesulfonyloxymethane)phosphonate and sodium tert-butoxide inDMF.¹⁴ Treatment of compound 19 with Me₃SiBr¹⁵ then afforded phosphonicacid 20 in 45% yield.

[0064] 9-(2-Cyanoethyl)adenine 15. To a suspension of 57% NaH in mineraloil (0.962 g, 22.8 mmol) in dry DMF (100 mL) was added 10 (3.40 g, 24.9mmol) under nitrogen and the mixture was heated at 80° C. for 1.0 h. Asolution of 3-bromopropionitrile (2.81 g, 21.0 mmol) in DMF (5.0 mL) wasadded at 25° C. and the reaction was heated at 65° C. for 17 h. It wasthen diluted with EtOAc (250 mL) and 5% aqueous HCl solution (150 mL).The organic layer was separated and then washed with water (4×100 mL).It was dried over MgSO₄ (s) and concentrated under reduced pressure.Purification of the residue was carried out by use of columnchromatography (EtOAc/hexanes=7.5:2.5) to afford compound 15 (3.510 g,18.67 mmol) in 75% yield: mp 148-150° C.; R_(f)(hexanes/EtOAc=1:2) 0.38;UV (EtOH) λ_(max) 260 (ε 13,900); ¹H NMR (CD₃OD) δ 2.92 (t J=5.8 Hz, 2H,CH₂CN),3.43 (t, J=5.8 Hz, 2H, CH₂N), 8.35 (s, 1H, HC₂), 8.69 (s, 1H,HC₈); MS m/z 188 (M⁺).

[0065] 7-[(Methoxycarbonyl)methyl]adenine 16 and9-[(Methoxycarbonyl)methyl]-adenine 17. To a stirred solution containing15 (0.134 g, 1.00 mmol) and methyl iodoacetate (0.30 g, 1.5 mmol) in dryTHF (20 mL) was added a THF solution of LiTMP (2.8 mL, 1.2 mmol)dropwise under an argon atmosphere at −20° C. The reaction mixture waswarmed to 25° C. within 1.0 h; then it was stirred at room temperaturefor 15 h. The solution was partitioned between EtOAc (40 mL) and water(40 mL). The organic layer was dried over MgSO₄ (s) and concentratedunder reduced pressure. Purification of the residue by use of columnchromatography (EtOAc/hexanes=8:2) gave compound 17 (0.041 g, 0.20 mmol)in 20% yield. Further elution of the column with EtOAc/hexanes (9:1)afforded 16 (0.123 g, 0.594 mmol) in 60% yield.

[0066] For 16: mp 112-114° C.; R_(f)(hexanes/EtOAc=1:2) 0.16; UV (EtOH)λ_(max) 267 (ε 14,000); ¹H NMR (CD₃OD) δ 3.83 (s 3H, CH₃), 4.37 (s, 2H,H₂C_(1′)), 8.41 (s, 1H, HC₂), 8.75 (s, 1H, HC₈); MS m/z 207 (M⁺). Anal(C₈H₉N₅0₂) C, H, N; calcd (%): 46.37, 4.38 33.80; found (%/o): 46.32,4.35, 33.83.

[0067] For 17: mp 148-149° C.; R_(f)(hexanes/EtOAc=1:2) 0.29; UV (EtOH)λ_(max) 260 (ε 13,750); ¹H NMR (CD₃OD) δ 3.75 (s, 3H, CH₃), 4.15 (s, 2H,H₂C_(1′)), 8.29 (s, 1H, HC₂), 8.38 (s, 1H, HC₈); MS m/z 207 (M⁺).

[0068] 7-(2-Hydroxyethyl)adenine 18. To a stirred solution containing 16(0.207 g, 0.999 mmol) and water (0.50 mL) in THF (12 mL) was added NaBH₄(0.38 g, 10.0 mmol). After stirring for 4 h at 25° C., the reactionmixture was neutralized to pH=7.0 by use of 10% HCl aqueous solution.Solvent was evaporated under reduced pressure and the residue waspurified by column chromatography (EtOAc/hexanes=9:1) to give 18 (0.10g, 0.55 mmol) in 55% yield: mp 132-133° C.; Rf(hexanes/EtOAc=1:2) 0.09;UV (EtOH) λ_(max) 265 (ε 14,110); ¹H NMR (CD₃OD) δ 3.68 (t, J=5.9 Hz,2H, CH₂O), 3.66 (t, J=5.9 Hz, 2H, CH₂N), 8.34 (s, 1H, HC₂), 8.68 (s, 1H,HC₈); MS m/z 179 (M⁺). Anal (C₇H₉N₅O) C, H, N; calcd (%): 46.92,5.06,39.08; found (%): 47.01,5.12, 39.11.

[0069] 7-[2-(D)iethylphosphonomethoxy)ethyl]adenine 19. To a solution of18 (0.18 g, 0.10 nmmol) in DMF (15 mL) was added sodium tert-butoxide(0.150 g, 1.56 mmol). After 5 min, diethyl(p-toluenesulfonyloxymethane)phosphonate (0.42 g, 1.3 mmol) was addedand the reaction mixture stirred at 35° C. for 4.0 h. The reaction wasthen quenched with acetic acid (5.0 mL) and the mixture was partitionedbetween EtOAc (50 mL) and water (50 mL). The organic layer was separatedand then washed with water (5×60 mL), dried over MgSO₄ (s), filtered,and concentrated under reduced pressure. Purification by use of columnchromatography (EtOAc/MeOH=9:1) gave 19 (0.20 g, 0.60 mmol) in 60% yieldas a white foam: R_(f)(hexanes/EtOAc=1:2) 0.16; UV (EtOH) λ_(max) 266 (ε15,321); ¹H NMR (CD₃OD) δ 1.39 (t, J=6.7 Hz, 6H, 2 CH₃), 3.74 (t, J=6.3Hz, 2H, CH₂N) 3.76 (d, J=9.0 Hz, 2H, CH₂P), 3.97-4.29 (m, 6H, CH₂O+2CH₂OP), 8.37 (s, 1H, HC₂), 8.77 (s, 1H, HC₈); MS m/z 329 (M⁺). Anal(C₁₂H₂₀N₅O₄P) C, H, N; calcd (%): 43.76, 6.12, 21.27; found (%): 43.87,6.21, 21.36.

[0070] 7-[2-(Phosphonomethoxy)ethyl]adenine 20 To a solution of 19 (3.29g, 10.0 mmol) in CH₂Cl₂ (130 mL) and DMF (10 mL) was added Me₃SiBr (4.95g, 30.0 mmol); then the solution was stirred at 25° C. for 7.0 h. Amixture of MeOH and water (5:1, 40 mL) was added, and solvents wereevaporated. Purification by use of column chromatography(EtOAc/MeOH=6:4) afforded 20 (1.23 g, 4.50 mmol) in 45% yield: mp 253°C. (dec.); R_(f) (hexanes/EtOAc=1:2) 0.05; UV (EtOH) λ_(max) 265 (ε14,700); ¹H NMR (CD₃OD) δ 3.66 (t, J=6.4 Hz, 2H, CH₂N), 3.71 (d, J=8.7Hz, 2H, CH₂P), 4.19 (t, J=6.4 Hz, 2H, CH₂O), 8.36 (s, 1H, HC₂), 8.78 (s,1H, HC₈); MS m/z 273 (M⁺). Anal (C₈H₁₂N₅O₄P) C, H, N; calcd (%): 35.17,4.43, 25.63; found (%): 35.21, 4.41, 25.71.

EXAMPLES 4-7 Synthesis of Compound 24, 25, 28, and 29

[0071]

[0072] As shown in Schemes 5 and 6, the nucleotide analog 24 was readilyobtained in three steps from adenosine 5′-monophosphate 21, startingwith silylation of phosphate 21 in CH₃CN with t-BuMe₂SiCl in thepresence of AgNO₃ and pyridine. See, e.g., Ogilvie et al. (1983) Can. J.Chem. 61: 1204-1212. The resulting trisilylated compound 22 wascondensed with phosphonic acid 20 using trichloromethanesulfonylchloride in collidine and THF to afford dinucleotide 5′-monophosphate 23in 42% overall yield. See, e.g., Hakimelahi et al. (1995) J. Med. Chem.38: 4648-4659 and references cited therein. Desilylation of 23 withn-Bu₄NF in THF at 25° C. gave dinucleotide 24 in 90% yield which wasthen reacted with(Z)-4-(2-chloroethylidene)-2,3-dimethoxy-Δ^(a,b)-butenolide (26) in thepresence of NaHCO₃ in DMF to afford the target molecule 25 in 85% yield(Hakimelahi et al. (2001) J. Med. Chem. 44: 1749-1757). Similarly,treatment of compound 26 with either9-[2-(phosphonomethoxy)ethyl]adenine (PMEA 4) or9-[2-(phosphonomethoxy)ethyl]guanine (PMEG 27) in the presence of NaHCO₃in DMF respectively gave an 80% or 88% yield of the desired compound 28or 29.

[0073]9-[2′-O-(tert-butyldimethylsilyl)-5′-O-(phosphono)-β-D-furanosyl]adenine-3′-[[1-(adenin-7-yl-ethoxy)methyl]phosphonate]23. To a solution of adenosine 5′-monophosphate (21) monohydrate (3.65g, 9.99 mmol) in a mixture of pyridine (150 mL) and CH₃CN (160 mL) wasadded AgNO₃ (6.63 g, 39.0 mmol). After 10 min, tert-butyldimethylsilylchloride (5.70 g, 37.8 mmol) was added. The mixture was stirred at 25°C. for 7.0 h and then filtered to remove AgCl. The filtrate wasevaporated and the resultant crude product 22 was dissolved in dry THF(40 mL). In another flask, collidine (3.66 g, 30.0 mmol) was added to asolution of THF (45 mL) containing 20 (2.73 g, 10.0 mmol) at −10° C. Tothis solution was added CCl₃SO₂Cl (2.20 g, 10.0 mmol) in THF (15 mL)dropwise. After crude 22 in THF was added to the mixture, it was stirredat 25° C. for 10 h. The solvents were removed, and the residue wasdissolved in AcOEt (100 mL) and washed with water (3×100 mL). Theorganic layer was concentrated, and the residue was purified by use ofcolumn chromatography (EtOAc/MeOH=6:4) to afford 23 (3.0 g, 4.2 mmol) in42% overall yield: mp 223-225° C.; UV (EtOH) λ_(max) 264 (ε 17,300); ¹HNMR (CD₃OD) δ 0.16 (brs, 6H, (CH₃)₂Si), 1.05 (s, 9H, (CH₃)₃C), 3.67-4.27(m, 8H, CH₂N+CH₂O+CH₂OP+CH₂P), 4.32-4.5 (m, 3H,HC_(2′)+HC_(3′)+HC_(4′)), 6.58 (d, J=4.9 Hz, 1H, HC_(1′)), 8.12, 8.42(2s, 2H, 2×HC₂), 8.27, 8.89 (2s, 2H, 2×HC₈). Anal (C₂₄H₃₈N₁₀P₂Si) C, H,N; calcd (%) 40.22, 5.34, 15.94; found (%): 40.29, 5.38, 19.50.

[0074]9-[5′-O-(phosphono)-β-D-furanosyl]adenine-3′-[[1-(adenin-7-yl-ethoxy)methyl]phosphonate]24. To a solution of 23 (0.36 g, 0.50 mmol) in THF (5.0 mL) was addedn-Bu₄NF (1.0 M solution in THF, 0.31 g, 1.2 mmol). Acetic acid (0.50 mL)was added to the mixture after it was stirred at 25° C. for 30 min. Thesolvents were removed, and the residue was purified by use of Whatman3-mm paper with a mixture of i-PrOH, NH₄OH, and H2O (9:1:2) as theeluent. The band at ca. R_(f) 0.35 was eluted with H₂O and collected bylyophilization to give 24 (0.27 g, 0.45 mmol) in 90% yield: mp>250° C.dec; UV (EtOH) λ_(max) 264 (ε 18,200); ¹H NMR (CD₃OD) δ 3.75-4.18 (m,8H, CH₂N+CH₂O+CH₂OP+CH₂P), 4.29-4.70 (m, 3H, HC_(2′)+HC_(3′)+HC_(4′)),6.48 (d, J=4.5 Hz, 1H, HC_(1′)), 7.99, 8.39 (2s, 2H, 2×HC₂), 8.26, 8.83(2s, 2H, 2×HC₈). Anal (C₁₈H₂₄N₁₀O₁₀P₂) C, H, N; calcd (%): 35.88, 4.02,23.25; found (%): 35.82, 4.12, 23.17.

[0075]9-[[(Z)-4-(ethylidene)-2,3-dimethoxy-Δ^(a,b)-butenolide]-β-D-furanosyl]adenine-3′-[[1-(adenin-7-yl-ethoxy)methyl]phosphonate]-4,5′-phosphate25. To a solution of 24 (0.300 g, 0.499 mmol) in DMF (20 mL) was addedNaHCO₃ (0.30 g, 3.6 mmol). The reaction mixture was stirred at 25° C.under N₂ for 10 min. Then, butenolide 26 (0.10 g, 0.50 mmol) was addedand stirred under N₂ for 1.0 h. The solution was diluted with EtOAc (50mL) and aqueous HCl solution (1%, 40 mL). The organic layer wasseparated and washed with H₂O (50 mL). Then, it was dried over MgSO₄(s), filtered, and concentrated under reduced pressure. Purification byuse of silica gel column chromatography with EtOAc/MeOH (6:4) as eluantafforded 25 (0.32 g, 0.42 mmol) in 85% yield: mp>237° C. dec; UV (EtOHl)λ_(max) 215 (ε 16,000), λ_(max) 264 (ε 18,540); ¹H NMR (CD₃OD) δ3.69-4.12 (m, 16H, CH₂N+CH₂O+2×CH₂OP+CH₂P+C₂OCH₃+C₃OCH₃), 4.31-4.78 (m,3H, HC_(2′)+HC_(3′)+HC_(4′)), 5.38 (t, J=7.0 Hz, 1H, ═CH), 6.51 (d,J=4.8 Hz, 1H, HC_(1′)), 8.02, 8.40 (2s, 2H. 2×HC₂), 8.28, 8.86 (2s, 2H.2×HC₈). Anal (C₂₆H₃₂N₁₀O₁₄P₂) C, H, N; calcd (%): 40.52, 4.19, 18.18;found (%): 40.61, 4.22, 18.21.

[0076][1-(Adenin-9-yl-ethoxy)methyl]phosphono-6-yl-(Z)-4-(ethylidene)-2,3-dimethoxy-Δ^(a,b)-butenolide28. Compound 28 (3.90 g, 8.80 mmol) was prepared in 88% yield from 4(2.73 g, 9.99 mmol) and 26 (2.20 g, 10.0 mmol) in the same manner that25 was prepared from 24: mp>241° C. (dec.); R_(f)(hexanes/EtOAc=1:2)0.12; UV (EtOH) λ_(max) 218 (ε 13,097), λ_(max) 259 (ε 14,700); ¹H NMR(CD₃OD) δ 3.57 (t, J=6.0 Hz, 2H, CH₂N), 3.69 (d, J=9.0 Hz, 2H, CH₂P),3.89 (m, 5H, C₂OCH₃+CH₂OP), 4.06, (t, J=6.0 Hz, 2H, CH₂O), 4.13 (s, 3H,C₃OCH₃), 5.41 (t, J=7.0 Hz, 1H, ═CH), 8.12 (s, 1H, HC₂), 8.21 (s, 1H,HC₈); MS m/z 441 (M⁺). Anal (C₁₆H₂₀N₅O₈P) C, H, N; calcd (%): 43.54,4.57, 15.86; found (%/o): 43.66, 4.46, 15.95.

[0077][1-(Guanine-9-yl-ethoxy)methyl]phosphono-6-yl-(Z)-4-(ethylidene)-2,3-dimethoxy-Δ^(a,b)-butenolide29. Compound 29 (3.7 g, 8.0 mmol) was prepared in 80% yield from 27(2.89 g, 9.99 mmol) and 26 (2.20 g, 10.0 mmol) in the same manner that25 was prepared from 24: mp>260° C. (dec.); R_(f)(hexanes/EtOAc=1:2)0.05; WV (EtOH) λ_(max) 252 (ε 11,097), λ_(max) 273 (ε 8,100); ¹H NMR(CD₃OD) δ 3.71 (t, J=7.2 Hz, 2H, CH₂N), 3.79 (d, J=9.1 Hz, 2H, CH₂P),3.92 (m, 5H, C₂OCH₃+CH₂OP ), 4.10, (t, J=7.2 Hz, 2H, CH₂O), 4.18 (s, 3H,C₃OCH₃), 5.50 (t, J=6.8 Hz, 1H, ⊚CH), 8.76 (s, 1H, HC₈); MS m/z 457(M⁺). Anal. (C₁₆H₂₀N₅O₉P) C, H, N; calcd (%): 42.02, 4.41, 15.31; found(%): 42.14, 4.50, 15.25.

EXAMPLE 8 Enzyme Assays

[0078] For comparison, compounds 1, 9-[(2-hydroxy-ethoxy)methyl]guanine(acyclovir 2), 9-(β-D-arabinofaranosyl)adenine (ara-A 3),9-[2-(phosphonomethoxy)ethyl]adenine (PMEA, 4), and9-[2-(phosphonomethoxy)ethyl]guanine (PMEG 27) were either synthesizedor purchased from a commercial source.

[0079] 9-[(2-Hydroxyethoxy)methyl]adenine 1. Compound 1 was prepared byan standard procedure: see Davari (1989) Doctoral Thesis, School ofVeterinary Medicine, Shiraz University, Shiraz, Iran. Mp 198-199° C.;R_(f)(hexanes/EtOAc=1:2) 0.23; UV (EtOH) λ_(max) 259 (ε 14,000); ¹H NMR(CD₃OD) δ 3.62 (s, 4H, O(CH₂)₂O), 5.67 (s, 2H, H₂C_(1′)), 8.22 (s, 1H,HC₂), 8.27 (s, 1H, HC₈); ¹³C NMR (CD₃OD) δ 61.86 (CH₂OH), 72.08 (OCH₂)74.31 (C_(1′)), 119.98 (C₅), 143.14 (C₈), 150.90 (C₄), 153.74 (C₂),157.19 (C₆); MS m/z 209 (M⁺).

[0080] Lipophilicity and Solubility Tests. Lipophilicity and watersolubility were determined by the distribution between 1-octanol andwater according to the methods reported by Baker et al. (1978, J. Med.Chem. 21: 1218-1221). The results show in Table 1.

[0081] To determine lipophoilicity (Partition Coefficients), a solutionof each compound (10 mL) in phosphate buffer (0.10 M) possessing an UVabsorbance of 2.2-3.3 at 258-267 nm, for adenine, or at 270-290 nm, forguanine, was shaken with 1-octanol (10 mL) in a separatory funnel for1.5 h. The layers were separated and their concentrations weredetermined by an UV spectrophotometer. The partition coefficient wascalculated as P=[S]_(1-octanol)/[S]_(H) ₂ _(O). To determine solubility,each compound (70 mg) was agitated in a 25-mL volumetric flask withphosphate buffer (0.10 M, pH 6.8, 5.0 mL) for 20 h. This solution wasfiltered from undissolved solid through a sintered glass funnel (4.0-5.5mesh ASTM) and the concentration of the solution was determined by UVabsorbance.

[0082] The results show that adenine acyclic nucleosides 1 and 14 wereobserved to exhibit higher lipophilicity as well as water solubilitythan those exhibited by N⁷-guanine acyclic nucleoside 9, acyclovir 2,and ara-A 3. Phosphonate 20 and butenolide ester derivatives 25, 28, and29 also exhibited higher lipophilicity and water solubility as comparedto PMEA 4 or PMEG 27. On the other hand, even though the solubility ofnucleotide analog 24 in water was found to be higher than that of PMEA4, its lipophilicity was lower than that of 4. Furthermore, butenolideester derivative 25 showed higher lipophilicity as well as watersolubility with respect to the parent nucleotide 5′-monophosphate 24.

[0083] Kinetic Studies of Competitive Inhibition ofAdenosine Deaminaseby Acyclic Nucleosides and Nucleotides. The rates of deamination ofN⁹-alkylated adenine 1, ara-A, 3, PMEA 4, N⁷-alkylated adenine 14,phosphonate 20, dinucleotide 5′-monophosphate 24, its butenolide esterderivative 25, and PMEA-containing butenolide 28 in the presence of calfmucosal adenosine deaminase (ADA, EC 3.5.4.4) in buffer solutions weredetermined. See, e.g., Ogilvie et al. (1984) Can. J. Chem. 62: 241-252and references cited therein. Additionally, the inhibition studies onthese compounds were carried out based on the Kaplan method (Table 2).See, e.g., Moosavi-Movahedi et al. (1993) Int. J. Biol. Macromol. 15:125-129 and references cited therein. The results (Table 2) showed thatboth the N⁹- and N⁷-acyclic nucleosides 1 and 14 functioned as ADAsubstrates. The V_(max) of 14 was less than that of 1 by a factor of 4.Compounds 1, 14, and 24 showed competitive inhibition of ADA when ara-Awas used as a substrate. However, N⁷-isomer 14 was found to be moreefficient than the N⁹-isomer 1 and nucleotide analog 24 as an inhibitorof ADA. PMEA 4, acyclic nucleoside phosphonate 20, andnucleotide-containing butenolides 25 and 28 were neither a goodsubstrate nor an inhibitor of the enzyme. Nucleotide analog 24, however,was a substrate for ADA, but its V_(max) was about 95% less than that ofara-A 3. The slow rate of deamination of compound 24 by ADA may reflectthe lack of substrate activity of the acyclic nucleoside phosphonatemoiety therein in the active site of the enzyme.

[0084] Comparison of Phosphorylation of Nucleosides by HSV and Vero CellThymidine Kinases. Phosphorylation of nucleosides with HSV or Vero cellthymidine kinase was studied as described previously. See, e.g., Kelleret al. (1981) Biochem. Pharmacol. 30: 3071-3077 and references citedtherein. Results are summarized in Table 3. The rate of phosphorylationof N⁹-adenine acyclic nucleoside 1, acyclovir 2, ara-A 3, N⁷-guanineacyclic nucleoside 9, and N⁷-adenine acyclic nucleoside 14 as well as amixture of 2 and 14 (w/w=1:1), a mixture of 1 and 2 (w/w=1: 1), and amixture of 2 and 3 (w/w=1:1) in the presence of HSV or Vero cellthymidine kinase was determined and the results were compared with thoseof thymidine. It was observed that N⁷-guanine nucleoside 9, unlikeacyclovir, can be phosphorylated by both the HSV thymidine kinase andthe host cell kinase. On the other hand, these enzymes were found not tophosphorylate adenine nucleosides 1, 14, and ara-A 3; yet the N⁷-adeninenucleoside 14 induced a 2-fold increase on the rate of phosphorylationof acyclovir 2 (see Table 3). N⁹-adenine nucleosides 1 and 3, however,did not induce any increase on the rate of phosphorylation of 2. It isbelieved that the N⁷-adenine nucleoside 14 binds (Km=30 mM) to aspecific receptor at the active site of the enzyme to exhibit theobserved activatory property toward the HSV thymidine kinase.

[0085] Enzymatic Conversion of Acyclic Nucleoside Phosphonates andNucleotide 5′-Monophosphate Analog to Their Antivirally ActiveDiphosphates (Triphosphate Equivalents). Substrate affinities of acyclicnucleoside phosphonates 4, 20, 21, and dinucleotide analog 24 for PRPPsynthetase as well as their inhibitory effect against the enzyme wereevaluated according to the established procedures. See, e.g., Balzariniet al. (1991) J. Biol. Chem. 266: 8686-8689 and references citedtherein. Results are illustrated in Table 4. The assays were thenterminated after 4 h by the addition of MeOH. HPLC on an anion-exchangePartisphere column was used to analyze the formation of ATP 21pp,PMEApp, 4pp, 20pp, and 24pp.

[0086] The substrate affinity of N⁷-acyclic nucleoside phosphonate 20(Km=4.8 mM) for the enzyme was found to be 4-times less than that ofPMEA 4 (Km=1.5 mM). Similarly, the V_(max) for conversion of 20 to 20ppis at least 7-times lower than that for the conversion of 4 to 4pp. Onthe other hand, nucleotide analog 24 was found to be a better substratethan PMEA 4 for PRPP synthetase (Km=0.57 mM). It can also bephosphorylated (V_(max)=12) by the enzyme at a rate similar to that ofAMP 21 (V_(max)=14).

[0087] It has been hypothesized (Balzarini supra) that the primary aminogroup at the C-6 position of purines is essential for hydrogen bondingwith the enzyme. The NH₂ group in 20 is sterically hindered by anadjacent side chain at the N-7 position. As such, it cannot aseffectively interact with the active site of the enzyme when compared toPMEA 4, natural substrate AMP 21, or nucleotide analog 24.

[0088] Activity ofSnake Venom and Spleen Phosphodiesterases AgainstNucleotide Analogs. Snake venom phosphodiesterase (200 units) wasdissolved in tris(hydroxymethyl)aminomethane buffer (1.0 mL), which wasadjusted to pH 9.2 with 0.1 N HCl. The enzyme solution (0.10 mL) wasadded to the nucleotide 24 or 25 (0.70 mg) and the mixture was incubatedat 37° C. for 8 h. The solution was then applied to Whatman 3-mm paperas a band, which was developed with a mixture of i-PrOH, conc. NH₄OH,and H₂O (9:1:2). Degradation products or unreacted starting materialswere separated. Dinucleotide 5′- monophosphate 24 gave9-(β-D-furanosyl)adenine-3′-[[1-(adenin-7-yl-ethoxy)methyl]phosphonate]in about 80% yield. On the other hand, dinucleotide-containingbutenolide 25 was completely resistant to the enzyme.

[0089] Spleen phosphodiesterase (20 units) was dissolved in sodiumpyrophosphate buffer (0.01 M, 1.0 mL), which was adjusted to pH 6.5 withphosphoric acid. Nucleotide 24, 25, or9-(β-D-furanosyl)adenine-3′-[[1-(adenin-7-yl-ethoxy)methyl]phosphonate](0.70 mg) was dissolved in ammonium acetate buffer (0.05 M, 0.20 mL),which was adjusted to pH 6.5 with acetic acid. An aliquot of the enzymesolution (0.1 mL) was added to the nucleotide solution and the mixturewas incubated at 37° C. for 8 h. The solution was then applied toWhatman 3-mm paper as a band and developed with a mixture of i-PrOH,conc. NH₄OH, and H₂O (9:1:2). Bands containing products were cut out,which were eluted with H₂O and the resultant mixture was freeze-dried.The isolated products were characterized by comparison with authenticsamples.

[0090] Dinucleotide 24 afforded phosphonate 20 and adenosine5′-monophosphate 21 in about 60% yield.9-(β-D-Furanosyl)adenine-3′-[[1-(adenin-7-yl-ethoxy)methyl]-phosphonate]gave adenosine (40% yield) and 20 (60% yield). Dinucleotide analog 25,having a butenolide ester unit, was found to be stable to theenzyme.Compound 24, possessing both the skeletons of phosphonate 20 and5′-adenosine monophosphate 21, was dephosphorylated at the 5′-positionby snake venom in 80% yield after 8 h. The resultant9-(b-D-furanosyl)adenine-3′-[[1-(adenin-7-yl-ethoxy)methyl]phosphonate]was hydrolyzed further in the presence of spleen phosphodiesterase toafford adenosine and phosphonate 20 in about 40-60% yield after 8 h.See, Hakimelahi et al. (1995) J. Med. Chem. 38: 4648-4659 and referencescited therein. Spleen phosphodiesterase also degraded compound 24 togive 5′-adenosine monophosphate 21 and phosphonate 20 in an overallyield of 60% after 8 h. These results indicate that thephosphodiesterases recognized dinucleotide analog 24 as a normalsubstrate. In addition, it was found that nucleotide-containingbutenolide 25 was completely resistant to snake venom and spleenenzymes.

EXAMPLE 9 In Vitro Assays

[0091] Anti-DNA Virus Activity in Vitro. The newly synthesized compoundswere tested for inhibition of cytopathogenicity of the herpes simplextype 1 virus (HSV-1), herpes simplex type 2 virus (HSV-2), thymidinekinase-positive (TK⁺) and thymidine kinase-deficient (TK⁻) strains ofvaricella-zoster virus (VZV), and human cytomegalovirus in Vero cellculture up to a level as high as 128 mg/mL. Compounds tested includeN⁷-alkylated purines 7, 9, 12, 14, 20, as well as a mixture of 1 and 14(w/w=1:1), 2 and 14 (w/w=1:1), 3 and 14 (w/w=1:1) in addition toN⁹-substituted adenine 1, acyclovir 2, ara-A 3, PMEA 4, PMEG 27,nucleotide analog 24, and nucleotide-containing butenolides 25, 28, and29. Toxicity of these compounds was evaluated by their ability to causemorphological changes in HeLa and Vero cells at differentconcentrations. The minimum inhibitory concentrations (IC₅₀) weremeasured by use of the linear regression method. See, e.g., Armitage(1983) “Statistical Methods in Medical Research,” Blackwell ScientificPublications, Oxford, U.K.; and Hakimelahi et al. (1990) J. Sci. Iran 1:186-191. The results are summarized in Table 5.

[0092] The pronounced anti-DNA virus activity of 14 with respect to thecorresponding chloro derivative 12 showed that the presence of ahydroxyl group is essential for antiviral activity. Results from thebiological tests also indicated that adenine nucleoside 14 was noteffectively deaminated by ADA; yet it inhibited the deactivatingproperty of the enzyme and led to the observed increase in the antiviralactivity of 1 and ara-A 3. Furthermore, use of adenine acyclicnucleoside 14 resulted in a 2-fold increment in the rate ofphosphorylation of acyclovir 2 by HSV-thymidine kinase. As such, acombination of 14 and 2 exhibited profound antiviral activity.N⁷-Adenine nucleoside 14 was found to be less toxic than thecorresponding N⁹-isomer 1. On the other hand, both HSV and cellularthymidine kinases can phosphorylate N⁷-guanine nucleoside 9. As aresult, this nucleoside exhibited more toxicity than acyclovir 2.

[0093] The rate of phosphorylation of N⁷-acyclic nucleoside phosphonate20 to its antivirally active anabolite 20pp by PRPP synthetase is7-times less than that of the PMEA 4. Thus in comparison to compound 4,compound 20 exhibited less activity against DNA-viruses. On the otherhand, nucleotide 5′-monophosphate analog 24, possessing a natural AMPmoiety, was converted to its diphosphate (triphosphate form) 24pp at arate comparable to that of AMP 21, which is about 120-times faster thanthe rate of conversion of PMEA 4 to PMEApp 4pp. Consequently, nucleotide5′-monophosphate 24 exhibited higher anti-DNA virus activity than PMEA4.

[0094] The ability of a drug to penetrate a membrane and exhibitbiological activity can be correlated to its lipophilicity. See, e.g.,Hakimelahi et al. (1995). J. Med. Chem. 38: 4648-4659 and referencescited therein. Consequently, compounds 25, 28, and 29 possessedbutenolide ester functionalities as lipophilic prodrugs. These compoundsdisplayed superior antiviral activity relative to their respectiveparent compounds nucleotide 5′-monophosphate 24, PMEA 4, and PMEG 27. Inaddition, spleen phosphodiesterase can recognize and at least partlyhydrolyze nucleotide analog 24 to the biologically less activephosphonate 20 inside the infected cells; whereas its butenolide esterderivative 25 was found to be stable toward phosphodiesterases. Thus incomparison with nucleotide analog 24, its prodrug 25 possesses superiorbioavailability and greater stability both in vitro and in vivo.

[0095] Anti-Retrovirus Activity in Vitro. The methods for measuringviruses-induced cytopathogenicity in MT4 cells or CEM cells, as well asthe toxicity of the tested compounds towards MT4 and CEM cells have beendescribed previously. See, e.g., Averett (1989) J. Virol. Methods 23:263-276. Results are summarized in Table 6.

[0096] Compounds 4, 20, 24, 25, 27, 28, and 29 were tested forinhibition of cytopathogenicity against the human immunodeficiencyviruses HIV-1 (III-B) and HIV-2 (LAV-2) in MT4 cells. These compoundswere also screened for their antiviral activity against moloney murinesarcoma virus (MSV) in CEM cells in a cell-protection assay.³⁰ Toxicityof these compounds was evaluated by their ability to cause morphologicalchanges in MT4 or CEM cells at different concentrations. The minimuminhibitory concentrations (IC₅₀) were measured by the use of the linearregression method (Table 6).

[0097] In comparison to the rate of phosphorylation of PMEA 4 to PMEApp4pp by PRPP synthetase, the conversion of dinucleotide 24 to itsanabolically active form 24pp is 120 times faster; yet PMEA 4 exhibitedhigher activity than 24 as well as the butenolide ester derivative 25against retroviruses. Thus, the HIV and MSV reverse transcriptases mayhave higher affinity for PMEA 4 than dinucleotide analog 24. PMEA 4 wasalso found to be more active than its N⁷-isomer 20 against retroviruses.On the other hand, butenolide ester derivatives 28 and 29 displayedsuperior antiviral activity relative to their respective parentmolecules 4 and 27. Thus in comparison to PMEA 4 and PMEG 27, theirrespective lipophilic prodrugs 28 and 29 possess superiorbioavailability and greater anti-retrovirus activity. As shown in Scheme6, we believe that the oxygen of the methoxy group at the C-2 positionof the butenolide moiety is responsible for the ease of conversion ofthese novel prodrugs 28 and 29 to their corresponding potential drugsPMEA 4 and PMEG 27 inside the infected cells.

EXAMPLE 10 In Vivo Assays

[0098] Anti-HSV-1 Activity in Vivo and Determination of LD₅₀ forN⁷-Adenine Acyclic Nucleoside 14, Nucleotide-Containing Butenolide 25,and PMEA-Containing Butenolide 28 in Mice. Two-weeks-old NMRI mice(15-20 animals/group), weighing ca. 7 g each, were infected ip. with4×10⁴ units of HSV-1 (KOS). See, e.g., Kim et al. (1991) J. Med. Chem.1991, 34, 2286-2294. Compounds in Table 7 were administered ip. once aday for 6 consecutive days, starting 4 h postinfection. Percentage ofHSV- 1-infected mice without symptoms and those that were alive at day21 postinfection were observed (see Table 7). Deaths were recorded for21 days after infection.

[0099] Acyclovir 2, PMEA 4, V7-guanine acyclic nucleoside 9, N⁷-adenineacyclic nucleoside 14, N⁷-acyclic nucleoside phosphonate 20, nucleotide5′-monophosphate 24, and butenolide ester derivatives 25 and 28 wereevaluated for their inhibitory effect on HSV-1-induced mortality in NMR₁mice (Table 6). Butenolide derivative of PMEA, 28, appeared to be themost potent anti-HSV-1 agent in vivo, followed by nucleotide-containingbutenolide 25, nucleoside analog 14, nucleotide analog 24, acyclovir 2,PMEA 4, phosphonate 20, and nucleoside analog 9. Since compound 28 isless active in vitro against HSV-1 when compared to compounds 25 and 14respectively, the in vitro potency does not directly translate to invivo potency. These results confirmed previous fmdings. See, e.g., DeClercq et al. (1986) Nature (London) 323: 464-467; and Pauwels et al.(1988) Agents Chemother. 32: 1025-1030.

[0100] All compounds were administered intraperitoneally (ip., 100-250mg/kg/day) for 6 consecutive days. Compounds 2, 4, 14, 24, and 25 gavefull protection against HSV-induced mortality at the 150 mg/kg doselevel. The same level of protection was provided by compound 28 at adose of 100 mg/kg. Survival times of all treated groups were found to besignificantly different from the placebo treated control group (seeTable 7). The potent anti-HSV-I activity exhibited by compounds 2, 4,14, 24, 25, and 28 clearly demonstrated that they are taken upeffectively by cells to exert in vivo activity. None of the compoundswere toxic to the mice at the highest dose tested.

[0101] The LD₅₀ values of the most active compounds 14, 25, and 28 inmice were also determined. As such, N⁷-acyclic nucleoside 14 andbutenolide ester derivatives 25 and 28 were administered at differentdoses intraperitoneally. They did not show any toxicity up to aconcentration level as high as 400 mg/kg. All mice were controlled ingood conditions after six months of administration. Nevertheless, LD₅₀(ip.) values of 950 mg/kg, 675 mg/kg, and 710 mg/kg were determined for14, 25, and 28, respectively. Moreover, no discernible abnormality wasobserved in the histological appearance of the viscera of either thecontrol or tested groups of mice that received the drugs ip. (250mg/kg/day) for 10 days. Furthermore, there were no physiological changesin their cardiovascular or central nerve systems.

[0102] Inhibitory Effects of PMEA 4 and Its Butenolide Ester Derivative28 on MSV-Induced Tumor Formation in Vivo. The inhibitory effects of thecompounds 4 and 28 on the initiation of MSV-induced tumor formation andsurvival of MSV-induced mice (10-15 animals/group) were evaluated aspreviously described. See, e.g., Balzarini et al. (1993) AgentsChemother 37: 332-338 and references cited therein. Results aresummarized in Table 8.

[0103] Compounds 4 and 28 were evaluated for their inhibitory effect onMSV-induced tumor formation in NMR₁ mice (Table 8). The compounds wereadministered intraperitoneally (50 mg/kg/day) for two consecutive days.Prodrug 28 exhibited much higher anti-MSV activity than PMEA 4 in vivo.At a dose of 10 mg/kg/day, compound 28 prevented tumor formation in 60%of the MSV-infected mice whereas with compound 4 at the same dosagelevel, only 19% prevention was observed. In surviving animals treatedwith 28, about 2 g weight loss was observed. In the case of PMEA-treatedmice, the weight loss of the surviving animals was at least two timesmore. TABLE 1 Solubility and Lipophilicity of Nucleoside and NucleotideAnalogs solubility in log P compound water (mg/mL) (1-octanol/water)^(a) 1 2.56 0.98 acyclovir 2 0.40 −0.48 ara A 3 0.48 −0.50 PMEA 4 1.97 0.69 9 0.32 −0.60 14 4.71 1.24 20 2.89 0.95 24 3.08 0.12 25 5.16 1.37 PMEG27 0.36 0.14 28 7.42 2.09 29 2.05 0.79

[0104] TABLE 2 Substrate Activity and Inhibitory Property AgainstADA^(a) Substrate K_(m) (□M) rel. V_(max) K_(i) (μM)  1 138.6 1.48 ×10⁻² 140.8 ara A 3 42.8 1.0 — PMEA 4 427.0 — >800 14 198.5 1.50 × 10⁻⁶8.3 20 >800 — >800 24 164.5 9.78 × 10⁻² 99.7 25 635.7 — >800 28 >800 —>800

[0105] TABLE 3 Phosphorylation of Various Nucleosides and Thymidine withHSV or Vero Cell Thymidine Kinases^(a) HSV thymidine kinase Vero cellthymidine kinase Substrate Km (□M) rel. Vmax Km μM) rel. Vmax  1 2.0 ×10⁴ <3.0 >3.0 × 10⁴ <3.0 acyclovir 2 1.5 39.2  2.2 × 10⁴ <3.0 ara A 31.5 × 10⁴ <3.0  1.3 × 10⁴ <3.0  9 12.8 28.0 18.3 12.0 14 30.0 <3.0 >3.0× 10⁴ <3.0  2 + 14 (1:1 w/w) — 80.9 — <3.0 thymidine 1.0 100.0 1.0 1.0 ×10²

[0106] TABLE 4 Kinetics of the PRPP Synthetase Reaction with AcyclicNucleoside Phosphonates 4 and 20, Nucleotide Analog 24, and AMP (21)^(a)substrate K_(m) (mM) V_(max) (μmol/unit/h) K_(i) ^(b) (mM) PMEA 4 1.510.096 3.02 20 4.83 0.013 16.74 AMP 21 0.24 14.270 — 24 0.57 11.560 0.79

[0107] TABLE 5 Anti-DNA Virus and Anticellular Activities of Nucleosideand Nucleotide Analogs in Tissue Culture. IC₅₀ ^(a) (μg/mL) HSV-1 HSV-2TK⁺VZV TK⁻VZV HCMV compound (KOS) (G) (YS) (YS/R) (AD-169) HeLa cell^(b)Vero cell^(b)  1 4.5 8.4 7.0 9.0 4.8 196 209 acyclovir 2 0.46 1.2 9.7 2832 259 269 ara A 3 8.8 >128 >128 >128 27 73 89 PMEA 4 11 7.4 6.1 6.0 15146 175  7 >128 >128 >128 >128 >128 157 145  9 1.1 2.8 13 38 16 114 9912 >128 >128 >128 >128 >128 169 165 14 0.58 0.97 1.1 0.97 1.0 472 496 2020 13 8.5 8.0 21 236 246 24 7.0 4.1 3.8 4.2 11 244 236 25 3.0 1.9 1.72.0 4.1 326 350 PMEG 27 4.6 6.3 0.040 0.060 0.35 9.1 7.0 28 3.7 3.0 2.02.4 5.4 139 170 29 0.42 0.28 0.0070 0.0080 0.020 8.8 6.6  1 + 14^(c)0.060 0.10 0.17 0.23 0.18 372 388  2 + 14^(c) 0.010 0.020 0.49 0.71 0.06420 410  3 + 14^(c) 0.10 1.1 0.98 1.7 0.46 280 293

[0108] TABLE 6 Inhibitory Effects of Nucleotide Analogs on theCytopathogenicity of HIV-1 and HIV-2 in MT4 Cells, as well as on theCytopathogenicity of MSV in CEM Cells and Cellular Toxicity. IC₅₀ ^(a)(μg/mL) MT4 CEM compound HIV-1 (IIIB) HIV-2 (LAV-2) MSV cell^(b)cell^(b) PMEA 4 4.1 3.8 2.0 274 285 20 7.8 9.1 27 >300 >300 24 5.9 6.417 298 >300 25 4.9 4.2 13 >300 >300 PMEG 27 16 18 0.19 16 12 28 1.4 1.00.93 265 280 29 6.0 7.1 0.020 14 13

[0109] TABLE 7 Antiviral Effects of compounds 2, 4, 9, 14, 20, 24, 25,and 28 Against HSV-1-Induced Mortality in NMRI Mice Upon IntraperitonealAdministration^(a) dose No. mean mean (mg/kg/ of day of symptom day ofanimal compound day) mice initiation (%)^(b) death (%)^(c) acyclovir 2250 20 >21 (100%) >21 (100%) 150 20 19.1 ± 1.3 (86%) >21 (100%) 100 1515.6 ± 1.6 (65%) 18.9 ± 2.1 (80%) PMEA 4 250 20 >21 (100%) >21 (100%)150 20 18.5 ± 1.9 (80%) >21 (100%) 100 15 14.7 ± 1.4 (56%) 17.0 ± 1.1(77%)  9 250 20 15.1 ± 1.2 (67%) 18.5 ± 1.0 (90%) 150 20 13.0 ± 1.1(57%) 16.0 ± 1.5 (78%) 100 15 10.9 ± 0.6 (48%) 14.1 ± 0.8 (66%) 14 25020 >21 (100%) >21 (100%) 150 20 19.9 ± 1.3 (88%) >21 (100%) 100 15 17.5± 2.1 (80%) 19.8 ± 1.7 (95%) 20 250 20 16.8 ± 2.4 (75%) 20.0 ± 1.7 (95%)150 20 15.0 ± 0.9 (62%) 17.8 ± 1.5 (80%) 100 15 12.8 ± 1.1 (51%) 15.4 ±1.3 (70%) 24 250 20 >21 (100%) >21 (100%) 150 20 19.3 ± 1.6 (85%) >21(100%) 100 15 16.0 ± 1.7 (70%) 19.5 ± 1.2 (90%) 25 250 20 >21 (100%) >21(100%) 150 20 >21 (100%) >21 (100%) 100 15 19.6 ± 1.5 (94%) >21 (100%)28 250 20 >21 (100%) >21 (100%) 150 20 >21 (100%) >21 (100%) 100 15 >21(100%) >21 (100%) Saline 0 20 3.38 ± 0.7 (0%) 9.4 ± 0.6 (0%)

[0110] TABLE 8 Inhibitory Effects of Acyclic Nucleoside Phosphonates 4and Its Prodrug 28 on MSV-Induced Tumor Formation and Associated Deathin NMRI Mice Upon Intraperitoneal Administration^(a) dose No. (mg/kg ofmean day of tumor mean day of animal compound day) mice initiation(%)^(b) death (%)^(c) PMEA 4 50 15 12.5 ± 1.6  (84%) 17.8 ± 2.0 (96%) 2015 12.0 ± 1.3  (58%) 15.9 ± 1.7 (76%) 10 10 9.6 ± 1.5  (19%) 13.0 ± 1.1(42%) 28 50 15 18.4 ± 1.4  (95%) >21 (100%) 20 15 17.9 ± 1.7  (80%) >21(100%) 10 10 14.0 ± 2.1  (60%) 18.9 ± 1.8 (97%) control 0 30 3.82 ± 0.95(0%) 7.8 ± 1.3 (0%) untreated control^(d) 0 40 >21 (100%) >21 (100%)

Other Embodiments

[0111] All of the features disclosed in this specification may becombined in any combination. Each feature disclosed in thisspecification may be replaced by an alternative feature serving thesame, equivalent, or similar purpose. Thus, unless expressly statedotherwise, each feature disclosed is only an example of a generic seriesof equivalent or similar features.

[0112] From the above description, one skilled in the art can easilyascertain the essential characteristics of the present invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usagesand conditions. Thus, other embodiments are also within the claims.

What is claimed is:
 1. A compound of formula (I):

wherein R₁ is NH₂ or OH; R₂ is H or NH₂; R₃ is H or alkyl; each of m andn, independently, is 1, 2, 3, or 4; X is O, S, or NH, and Y is H,halogen, OR^(a), P(O)(OR^(a))₂, or P(O)(OR^(a))(OR^(b)), in which R^(a)is H, alkyl, aryl, heteroaryl, cyclyl, heterocyclyl, and R^(b) is

being adenine, guanine, cytosine, uracil, or thymine, R^(c) being H orOH, R^(d) being H or alkyl, R^(e) being H, alkyl, or5-ethylidene-(3,4-dialkoxyl)-furan-2-one; provided that if R₁ is NH₂, R₂is H; and if R₁ is OH, R₂ is NH₂.
 2. The compound of claim 1, wherein R₁is NH₂ and R₂ is H.
 3. The compound of claim 2, wherein R₃ is H.
 4. Thecompound of claim 3, wherein m is
 1. 5. The compound of claim 4, whereinX is O and n is
 2. 6. The compound of claim 5, wherein Y is OR^(a). 7.The compound of claim 6, wherein R^(a) is H.
 8. The compound of claim 3,wherein m is
 2. 9. The compound of claim 8, wherein X is O and n is 1.10. The compound of claim 9, wherein Y is P(O)(OR^(a))₂.
 11. Thecompound of claim 10, wherein R^(a) is H.
 12. The compound of claim 9,wherein Y is P(O)(OR^(a))(OR^(b)).
 13. The compound of claim 12, whereinR^(a) is H and R^(b) is


14. The compound of claim 13, wherein each of R^(c), R^(d), and R^(e) isH.
 15. The compound of claim 13, wherein each of R^(c) and R^(d) is H,and R^(e) is 5-ethylidene-(3,4-dialkoxyl)-furan-2-one, in which thedialkoxyl is dimethoxyl.
 16. The compound of claim 2, wherein X is O.17. The compound of claim 2, wherein each of m and n, independently, is1 or
 2. 18. The compound of claim 1, wherein R₁ is OH and R₂ is NH₂. 19.The compound of claim 18, wherein R₃ is H.
 20. The compound of claim 19,wherein m is
 1. 21. The compound of claim 20, wherein X is O and n is 2.22. The compound of claim 21, wherein Y is OR^(a).
 23. The compound ofclaim 22, wherein R^(a) is H.
 24. The compound of claim 19, wherein m is2.
 25. The compound of claim 24, wherein X is O and n is
 1. 26. Thecompound of claim 25, wherein Y is P(O)(OR^(a))₂.
 27. The compound ofclaim 26, wherein R^(a) is H.
 28. The compound of claim 25, wherein Y isP(O)(OR^(a))(OR^(b)).
 29. The compound of claim 18, wherein X is O. 30.The compound of claim 18, wherein each of m and n, independently, is 1or
 2. 31. A method of treating infection by virus, comprisingadministering to a subject in need thereof an effective amount of acompound of formula (I):

wherein R₁ is NH₂ or OH; R₂ is H or NH₂; R₃ is H or alkyl; each of m andn, independently, is 1, 2, 3, or 4; X is O, S, or NH, and Y is H,halogen, OR_(a), P(O)(OR^(a))₂, or P(O)(OR^(a))(OR^(b)), in which R_(a)is H, alkyl, aryl, heteroaryl, cyclyl, heterocyclyl, and R^(b) is

A being adenine, guanine, cytosine, uracil, or thymine, R^(c) being H orOH, R^(d) being H or alkyl, R^(e) being H, alkyl, or5-ethylidene-(3,4-dialkoxyl)-furan-2-one; provided that if R₁ is NH₂, R₂is H; and if R₁ is OH, R₂ is NH₂.
 32. The method of claim 3 1, whereinR₁ is NH₂ and R₂ is H.
 33. The method of claim 31, wherein R₁ is OH andR₂ is NH₂.
 34. The method of claim 3 1, wherein the virus is DNA virus.35. The method of claim 34, wherein the virus is herpesvirus.
 36. Themethod of claim 35, wherein the virus is herpes simplex virus.
 37. Themethod of claim 31, wherein the virus is retrovirus.
 38. The method ofclaim 37, wherein the virus is human immunodeficiency virus.
 39. Themethod of claim 37, wherein the virus is moloney murine sarcoma virus.40. A compound of formula (II):

wherein R₁ is NH₂ or OH; R₂ is H or NH₂; R₃ is H or alkyl; each of m andn, independently, is 1, 2, 3, or 4; X is O, S, or NH, and Y isP(O)(OR^(a))(OR^(b)), in which R^(a) is H, alkyl, aryl, heteroaryl,cyclyl, heterocyclyl, and R^(b) is5-ethylidene-(3,4-dialkoxy)-furan-2-one or

A being adenine, guanine, cytosine, uracil, or thymine, R^(c) being H orOH, R^(d) being H or alkyl, R^(e) being H, alkyl, or5-ethylidene-(3,4-dialkoxyl)-furan-2-one; provided that if R₁ is NH₂, R₂is H; and if R₁ is OH, R₂ is NH₂.
 41. The compound of claim 40, whereinR₁ is NH₂ and R₂ is H.
 42. The compound of claim 41, wherein R₃ is H.43. The compound of claim 42, wherein m is
 2. 44. The compound of claim43, wherein X is O and n is
 1. 45. The compound of claim 44, whereinR^(a) is H and R^(b) is 5-ethylidene-(3,4-dialkoxy)-furan-2-one.
 46. Thecompound of claim 41, wherein X is O.
 47. The compound of claim 41,wherein each of m and n, independently, is 1 or
 2. 48. The compound ofclaim 40, wherein R₁ is OH and R₂ is NH₂.
 49. The compound of claim 48,wherein R₃ is H.
 50. The compound of claim 49, wherein m is
 2. 51. Thecompound of claim 50, wherein X is O and n is
 1. 52. The compound ofclaim 51, wherein R_(a) is H and R^(b) is5-ethylidene-(3,4-dialkoxy)-furan-2-one.
 53. The compound of claim 48,wherein X is O.
 54. The compound of claim 48, wherein each of m and n,independently, is 1 or
 2. 55. A method of treating infection by virus,comprising administering to a subject in need thereof an effectiveamount of a compound of formula (II):

wherein R₁ is NH₂ or OH; R₂ is H or NH₂; R₃ is H or alkyl; each of m andn, independently, is 1, 2, 3, or 4; X is O, S, or NH, and Y isP(O)(OR^(a))(OR^(b)), in which R^(a) is H, alkyl, aryl, heteroaryl,cyclyl, heterocyclyl, and R^(b) is5-ethylidene-(3,4-dialkoxy)-furan-2-one or

A being adenine, guanine, cytosine, uracil, or thymine, R^(c) being H orOH, R^(d) being H or alkyl, R^(e) being H, alkyl, or5-ethylidene-(3,4-dialkoxyl)-furan-2-one; provided that if R₁ is NH₂, R₂is H; and if R₁ is OH, R₂ is NH₂.
 56. The method of claim 55, wherein R₁is NH₂ and R₂ is H.
 57. The method of claim 55, wherein R₁ is OH and R₂is NH₂.
 58. The method of claim 55, wherein the virus is DNA virus. 59.The method of claim 58, wherein the virus is herpesvirus.
 60. The methodof claim 59, wherein the virus is herpes simplex virus.
 61. The methodof claim 59, wherein the virus is retrovirus.
 62. The method of claim61, wherein the virus is human immunodeficiency virus.
 63. The method ofclaim 62, wherein the virus is moloney murine sarcoma virus.
 64. Amethod comprising reacting a compound of formula (III):

with an alkyl-X-(CH₂) halide to obtain a compound of formula (IV):

in which R₁ is NH₂ or OH; R₂ is H or NH₂; R₃ is H or alkyl; and X is O,S, or NH; provided that if R₁ is NH₂, R₂ is H; and if R₁ is OH, R₂ isNH₂.
 65. The method of claim 66, wherein R₁ is NH₂ and R₂ is H.
 66. Themethod of claim 66, wherein R₁ is OH and R₂ is NH₂.
 67. The method ofclaim 66, wherein X is O.
 68. The method of claim 66, wherein R₃ is H.